Abstract: :
Purpose: Glutathione S-transferases (GSTs) are a large groupof enzymes divided into two superfamilies; one soluble (e.g.alpha, mu, pi classes) and the other membrane-bound (e.g. microsomalI class). An important physiological function of GSTs is todetoxify the cell from reactive metabolites generated in thecourse of normal metabolism and in pathology. Current researchis paying particular attention to GST-induced suppression ofapoptosis. As a result the objective of our studies is to seeif GSTs rescue photoreceptors in rd/rd mouse retinal explants(RE). GST-mu was investigated because the mu and alpha classesare present in rat retinal elements that are linked to photoreceptorrescue.Methods: Eyes from C3H rd/rd and +/+ mice were collectedat postnatal day (PN) 2, PN7, PN14, PN21 and PN28. From eachage group some eyes were fixed with 4% paraformaldehyde, 10µm-frozensections were cut and incubated with a polyclonal antibody directedagainst GST-mu (1:500, Oxford Biomedical Research). The boundprimary antibodies were detected using immunofluorescence. Fromother eye bulbs the retina was isolated, total protein extracted,separated using SDS-PAGE and proteins transferred to a nitrocellulosemembrane. These blots were incubated with the GST-mu antibody(1:500) and the bound antibody detected through chromagen orchemiluminescence. PN2 and PN7 rd/rd and +/+ RE were generatedas previously described and incubated with and without 10ng/mlGST-mu up to PN28. In RE sections the number of surviving photoreceptorswas analyzed statistically.Results: From PN2 onwards GST-muwas present in Müller cells in both rd/rd and +/+ retina.Western blot analyses detected one protein band of around 26kDa which further indicated that GST-mu protein levels in therd/rd were lower than those in the +/+ mouse retina. The numberof rows of photoreceptors in PN2 rd/rd RE exposed to GST-muwas 6 while PN7 RE cultured in similar conditions showed only3 rows. In both PN2 and PN7 rd/rd RE without GST-mu, 2 to 3rows of photoreceptor cells were present. In +/+ RE with andwithout GST-mu, 7 to 8 rows were observed.Conclusion: In mouseretina GST-mu is present early in development and localizedto Müller cells. The lower GST-mu protein levels in therd/rd retina might contribute to cell loss. When exogenous GST-muis applied this treatment rescues photoreceptors only in PN2rd/rd RE. In this model the protective potency of GST-mu issimilar to that of CNTF alone and NGF+FGF2. Further study needsto establish if other members of the GST superfamily rescuerd/rd photoreceptors as well.